Alzheimer's disease (AD) is an irreversible, progressive neurodegenerative disease that slowly destroys memory and thinking skills, and eventually causes senile dementia. More than 5 million people in the U.S. and 24 million people worldwide suffer from this disease. The pathogenesis of AD is far from being understood, and 42- and 40-amino acids long amyloid β peptides (Aβ42 and Aβ40, respectively) are proposed to play a central role in the onset of AD. Although Aβ40 is present in larger amounts in the brain, Aβ42 was found to be more neurotoxic and has a higher tendency to aggregate. The amyloid cascade hypothesis suggests the ultimate products of Aβ aggregation, the amyloid plaques, are responsible for neurodegeneration. However, recent in vivo studies have shown that soluble Aβ oligomers are more neurotoxic than amyloid plaques and most likely responsible for synaptic dysfunction and memory loss in AD (Sharma et al., Metallomics, 2013, 5: 1529-1536).
The development of effective treatments for AD has been hindered, in large part, by the lack of methods available to definitively diagnose AD in living patients. Although AD is routinely diagnosed clinically after a series of mental status tests and physical examination, it can only be confirmed by demonstrating that the pathologic hallmarks of AD-protein deposits and neurofibrillary tangles are present in the brain. Major neuropathology observations of postmortem examination of AD brains confirm the presence of AD through the detection of extracellular β-amyloid peptides and intracellular neurofibrillary tangles (NFT). NFTs derive from filaments of hyperphosphorylated tau proteins. The presence and severity of NTFs correlate with severity of dementia and cognitive impairment (Dickson, D. W., Neurobiology of Aging, 1997, 18(4): S21-S26). The presence of these deposits is typically confirmed post mortem through autopsy of the brain. Autopsied brain tissues are treated with histologic dyes, such as Thioflavin T (ThT) or Congo Red (CR), which have an affinity for and stain amyloid plaques. These dyes are limited to post mortem use because their inherent charges prevent them from crossing the blood brain barrier in vivo.
In 2002, a labeled derivative of Thioflavin T known as 11C-Pittsburgh Compound Blue (11C-PiB) was developed at the University of Pittsburgh. 11C-PiB was the first PET agent to show success in clinical trials, and is now the most well-characterized and studied radiopharmaceutical for Aβ pathology. In vitro autoradiography studies have confirmed that 11C-PiB binds to aggregated, fibrillar Aβ deposits in the cortex, striatum, and cerebral vessel walls, but not to amorphous, cerebellar Aβ deposits. At nanomolar concentrations, however, 11C-PiB does not bind to free soluble amyloid, tau neurofibrillary tangles (NFTs), or Lewy bodies (Zeman et al., 12 Chapters on Nuclear Medicine, 2011: 199-230).
Routine clinical use of 11C-PiB is limited by the short half-life of the 11C radioisotope (20.4 min), which necessitates on-site synthesis and immediate use of 11C-PiB for PET scanning. As a result, Aβ PET tracers that are radiolabeled with fluorine-18, a radioisotope with a considerably longer half-life (110 minutes) than carbon-11, have been developed. Fluorine-18 labeled Aβ PET tracers do not require on-site cyclotrons for their production, thus allowing for a more widespread distribution of this imaging technology. Several 18F-labeled Aβ PET radiopharmaceuticals have been developed, including 18F-flutemetamol, 18F-florbetaben, 18F-florbetapir, and 18F-AZD4694 (also named 18F-NAV4694). One such compound, 18F-forbetapir, was approved by the U.S. Food and Drug Administration in April 2012. The structures of the five Aβ PET radiopharmaceuticals in use at multiple sites to image Alzheimer pathology in vivo are shown below.

Recently, Donnelly et al. reported the development of copper radiopharmaceuticals that target Aβ plaques for non-invasive diagnosis and monitoring of amyloid diseases (J. Am. Chem. Soc., 2013, 135:16120-16132). Initial attempts focused on derivatives of CuII(astm)2, a known hypoxia imaging agent. It was found that bis(thiocarbazone) ligands were difficult to radiolabel with Cu-64 and that the thiocarbazone linkage was susceptible to hydrolysis. Subsequently, hybrid ligands (CuIIL1, CuIIL2, and CuIIL3) based on thiosemicarbazone-pyridylhydrazone were synthesized, and shown to form more stable, charge neutral CuII complexes.

Benzothiazole 11C-PiB was the structural inspiration behind the preparation of ligand (H2L1) for metal complex CuIIL1. Unfortunately, copper complex CuIIL1 suffered from very poor solubility and did not appear to selectively interact with human AD post-mortem brain tissue. In contrast, copper complexes CuIIL2 and CuIIL3 that incorporated a styrylpyridyl group instead of a benzothiazole group were found to bind to Aβ plaque. However, in preliminary biodistribution studies of copper-64 radiolabeled complexes of CuIIL2 and CuIIL3 in normal mice, only CuIIL3 crossed the blood-brain barrier. Thus, subtle differences in the ligand framework altered both the binding of the metal complex to Aβ plaque and its ability to enter the central nervous system.
The present inventors previously reported the synthesis of bifunctional chelators that contain amyloid binding and metal chelating motifs (Sharma et al., J. Am. Chem. Soc., 2012, 134: 6625-6636). Thus, bifunctional chelators L1 and L2 were designed with a 2-arylbenzothiazole moiety to enable binding to Aβ and an N-(2-pyridylmethyl) amine group to simultaneously chelate transition metals such as Cu, Zn, or Fe that have been found within amyloid deposits in AD brain tissue.

Both compounds L1 and L2 were found to be efficient inhibitors of the metal-mediated aggregation of the Aβ42 peptide and promoted disaggregation of amyloid fibrils. However, the ability of L1 and L2 to inhibit Aβ fibril formation and promote fibril disaggregation led to increased cellular toxicity, especially for L2, limiting their potential as therapeutic agents.
There remains a need for improved imaging agents for the detection of Aβ species in Alzheimer's disease. Non-invasive functional molecular imaging techniques such as PET imaging have the potential to become the future diagnostic standard for Alzheimer's disease, as they would allow for earlier and more definitive diagnosis of such diseases and provide a more effective method for monitoring possible treatments. Thus, there is a need for nontoxic and longer-lived radiopharmaceutical compounds that bind selectively and with high affinity to various Aβ species, including β-amyloid plaques and neurofibrillary tangles.